Systematic identification of long noncoding RNAs during three key organogenesis stages in zebrafish

Abstract

Abstract Background Thousands of lncRNAs have been found in zebrafish embryogenesis and adult tissues, but their identification and organogenesis-related function have not elucidated. Results In this study, high-throughput sequencing was performed at three different organogenesis stages of zebrafish embryos, which were important for zebrafish muscle development. The three stages were 10 hpf (hours post fertilization) (T1), 24 hpf (T2) and 36 hpf (T3). LncRNA gas5 associated with muscle development was screened out as the next research target by high-throughput sequencing and qPCR validation. The spatiotemporal expression of lncRNA gas5 in zebrafish embryonic muscle development was studied by qPCR and in situ hybridization, and functional analysis was conducted using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/Cas9, CRISPR/Cas9). The results were as follows: 1) A total of 1486 differentially expressed lncRNAs were identified between 24 hpf (T2) and 10 hpf (T1), among which 843 lncRNAs were upregulated and 643 were downregulated. Compared with 36 hpf (T3) and 24 hpf (T2), a total of 844 differentially expressed lncRNAs were screened, among which 482 lncRNAs were upregulated and 362 lncRNAs were downregulated. Compared with 36 hpf (T3) and 10 hpf (T1), a total of 2137 differentially expressed lncRNAs were screened, among which 1148 lncRNAs were upregulated and 989 lncRNAs were downregulated, including lncRNA gas5, which was selected as the target gene. 2) The result of spatiotemporal expression analysis showed that lncRNA gas5 was expressed in almost all detected embryos of different developmental stages (0, 2, 6, 10, 16, 24, 36, 48, 72, 96 hpf) and detected tissues of adult zebra fish. 3) After lncRNA gas5 knockout using CRISPR/Cas9 technology, the expression levels of detected genes related to muscle development and adjacent to lncRNA gas5 were affected in the knockout group compared with the control group, suggesting that lncRNA gas5 may play a role in embryonic muscle development in zebrafish. Conclusion The result of this study provide an experimental basis for further study on the role of lncRNA gas5 in zebrafish embryonic muscle development.